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E M Callaway

Publications and source records attributed to E M Callaway.

9 recordsLinked to original sources
PubMed2009

Preferential labeling of inhibitory and excitatory cortical neurons by endogenous tropism of adeno-associated virus and lentivirus vectors.

Despite increasingly widespread use of recombinant adeno-associated virus (AAV) and lentiviral (LV) vectors for transduction of neurons in a wide range of brain structures and species, the diversity of cell types within a given brain structure is rarely considered. For example, the ability of a vector to transduce neurons within a brain structure is often assumed to indicate that all neuron types within the structure are transduced. We have characterized the transduction of mouse somatosensory cortical neuron types by recombinant AAV pseudotyped with serotype 1 capsid (rAAV2/1) and by recombinant lentivirus pseudotyped with the vesicular stomatitis virus (VSV) glycoprotein. Both vectors used human synapsin (hSyn) promoter driving DsRed-Express. We demonstrate that high titer rAAV2/1-hSyn efficiently transduces both cortical excitatory and inhibitory neuronal populations, but use of lower titers exposes a strong preference for transduction of cortical inhibitory neurons and layer 5 pyramidal neurons. In contrast, we find that VSV-G-LV-hSyn principally labels excitatory cortical neurons at the highest viral titer generated. These findings demonstrate that endogenous tropism of rAAV2/1 and VSV-G-LV can be used to obtain preferential gene expression in mouse somatosensory cortical inhibitory and excitatory neuron populations, respectively.

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PubMed1992

Development of axonal arbors of layer 4 spiny neurons in cat striate cortex.

Spiny neurons in layer 4 of cat striate cortex are the primary recipients of geniculocortical afferents and provide crucial links to other cortical layers for processing visual information. Using intracellular staining, we examined the development of the local axonal projections of these neurons to determine (1) whether the laminar specificity of their projections emerged specifically or was sculpted from transient exuberant projections and (2) whether the emergence of excitatory connections from layer 4 to layer 2/3 could contribute to the activity-dependent development of clustered horizontal connections of layer 2/3 pyramidal neurons. Differences in the extent of projections to infragranular (layers 5 and 6, which receive sparse projections) versus superficial layers (layers 2/3 and 4, which receive extensive projections) developed specifically from the outset. By postnatal day 15 (P15) projections to infragranular layers matured and were indistinguishable from those in the oldest animal studied (P33). In contrast, projections to superficial layers continued to increase in complexity after P15. Projections within layer 4, which were the most elaborate at all ages studied, reached maturity at about P20, while those to layer 2/3 continued to increase in complexity through P33. No evidence for exuberant projections to any of these cortical layers was observed. At very early postnatal ages (P5) projections to the subplate region were evident. These disappeared by P8-P11, suggesting the presence of transient connections from layer 4 spiny neurons to subplate neurons. Binocular deprivation did not prevent the emergence of projections from layer 4 spiny neurons into layer 2/3 or development of normal laminar differences in projection density. Connections from layer 4 to layer 2/3 emerged after horizontal connections in layer 2/3 were crudely clustered, but in synchrony with the later refinement of clusters. Collaterals from layer 4 cells first crossed into layer 3 at P11, but were extremely short (extending only 50-200 microns beyond the laminar boundary) and uncommon (only 4 of 19 cells). Since by P8 horizontal projections of layer 2/3 pyramidal neurons are already crudely clustered, the emergence of crude clustering is probably independent of layer 4 to layer 2/3 excitatory projections. The proportion of cells projecting to layer 2/3 and the complexity of their arbors both increased in the subsequent weeks, closely matching the timing of both the refinement of crudely clustered horizontal connections and the emergence of visual responsiveness in layer 2/3.(ABSTRACT TRUNCATED AT 400 WORDS)

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PubMed1991

Effects of binocular deprivation on the development of clustered horizontal connections in cat striate cortex.

Intrinsic horizontal axon collaterals in the striate cortex of adult cats specifically link columns having the same preferred orientation; consequently, retrograde tracer injections result in intrinsic labeling that is sharply clustered. We have previously shown that the normal development of this circuitry involves the emergence of crude clusters from an unclustered pattern during the second postnatal week. Crude clusters are later refined to the adult level of specificity by the selective rearrangement of axonal arbors that initially project to incorrect orientation columns. Here we report that depriving animals of patterned visual experience by binocular lid suture prior to natural eye opening had no discernible effect on the emergence of crude clusters. In contrast, cluster refinement was dramatically affected by binocular deprivation. Injections of retrograde tracers in the striate cortex of animals binocularly deprived for greater than 1 month revealed only crude clusters, indicating that horizontal axon collaterals projecting to incorrect orientation columns were retained well past the age when they normally would have been eliminated. Layer 2/3 pyramidal cells from 6-week-old binocularly deprived animals had abnormal distributions of intrinsic horizontal axon collaterals that mirrored the lack of cluster refinement. The radial clustering of their horizontal collaterals was considerably less precise than normal. These cells, nevertheless, developed many of the features of normal mature arbors, including the distal axonal branches not seen in arbors from younger animals with normal visual experience. Together, these results indicate that axonal rearrangements occurred, but with reduced specificity. Thus, binocular deprivation did not simply arrest the development of this orientation-specific circuit at an immature state but limited the accuracy with which axon collaterals were added or eliminated. We suggest that development of this orientation-specific circuitry, like ocular dominance column segregation, may depend on temporal correlation of activity for regulation of axonal rearrangement. The specificity of rearrangement may be degraded in binocularly deprived cats because they do not experience sharply oriented visual stimuli necessary for concurrent activation of same-orientation columns.

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PubMed1990

Emergence and refinement of clustered horizontal connections in cat striate cortex.

Pyramidal cells in layer 2/3 of adult cat striate cortex have long, intrinsic horizontal axon collaterals within both layer 2/3 and layer 5. These collaterals form periodic "clusters" of finer axon branches that link columns of similar orientation selectivity. We have investigated the sequence of events and possible mechanisms underlying the development of these clustered intrinsic horizontal connections using a combination of neuronal tracers and intracellular staining. Small injections of fluorescent latex microspheres made during the first postnatal week (at P4-6), when examined in tangential sections, produced an even, unclustered distribution of retrogradely labeled cells up to 2 mm from the injection site. At P8, retrograde labeling extended over a larger area and clustering was discernible, primarily among the most distant labeled cells. At both P6 and P8, labeling was similar in layers 2/3 and 5, indicating that the transition from clustered to unclustered connections occurred simultaneously for cells in superficial and deep laminae. By the end of the second postnatal week (P12-15), retrogradely labeled cells were far more clustered both within and beyond the extent of P6 label; the density of labeled cells was high throughout the labeled region, but much higher within clusters. The periodicity of these nascent clusters was similar to that in the adult. Despite obvious clustering, the pattern of retrograde label observed following injections at 2-3 weeks (P12-21) differed markedly from the adult, in that the regions between clusters contained many labeled cells. Over the next 3 weeks, the connections were refined, so that by the sixth postnatal week (P36-38), regions between clusters contained very few retrogradely labeled cells and the overall pattern of retrograde label was indistinguishable from that in adults. Despite differences in postmigratory ages of neurons from the superficial and deep laminae, clustering of retrogradely labeled cells from these 2 populations was similar at all ages. Experiments in which 2-3 weeks elapsed between the time microsphere injections were made and animals were killed demonstrated that neither the initial formation of crude clusters nor their refinement was due to cell death. Instead, cluster refinement resulted from specific process elimination. When a red microsphere injection at P15 was followed by a green microsphere injection at exactly the same location on P29, the earlier injection resulted in crude clustering, as expected. Virtually all of the cells double-labeled by the later injection were within the densest clusters of label from the early injection.(ABSTRACT TRUNCATED AT 400 WORDS)

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PubMed1989

Slowing of synapse elimination by alpha-bungarotoxin superfusion of the neonatal rabbit soleus muscle.

To examine the role of postsynaptic activity in regulating the rate of neuromuscular synapse elimination, contractile activity of neonatal rabbit soleus muscles was decreased by chronic superfusion of alpha-bungarotoxin (alpha-BGT) over their surfaces. Superfusion was begun at 6 days postnatal and continued for a variable duration (2 to 5 days) before muscles were analyzed. The percentage of polyinnervated fibers was assessed both physiologically and anatomically for alpha-BGT-treated muscles and their contralateral muscles, in addition to normal and control muscles of the same age. Within muscles exposed to alpha-BGT, polyinnervation was significantly greater than that for muscles from each of the control groups. The anatomical assay further revealed that the retention of polyinnervation in alpha-BGT-treated muscles was most pronounced near the muscle's surface, although end plates at the center were also affected. This finding, coupled with evidence that only a small percentage of the muscle fibers were completely inactivated, suggests that the activity block was also most pronounced near the surface and relatively low at the muscle's center. The percentage of end plates at which synapse elimination was delayed was greater than the estimated percentage whose activity was completely blocked, suggesting that synapse loss was slowed even in muscle fibers retaining some postsynaptic activity. These observations indicate that the rate of synapse elimination depends on the levels of functional acetylcholine receptors. This process could be mediated in a graded fashion by changes in postsynaptic activity (subthreshold or suprathreshold) or by a nonelectrical effect of blocking postsynaptic receptors.

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PubMed1989

Lack of fiber type selectivity during reinnervation of neonatal rabbit soleus muscle.

Fast and slow contracting fibers in neonatal mammalian skeletal muscle are each innervated in a highly specific manner by motor neurons of the corresponding type, even at an age when polyinnervation is widespread. Chemospecific recognition is a possible mechanism by which this pattern of innervation could be established. We have investigated this possibility by studying the degree of specificity during reinnervation of rabbit soleus muscle following nerve crush on Postnatal Day 1 or 4. We assayed fiber type composition by measuring the twitch rise times of motor units within 2 days of the onset of functional reinnervation (5-6 days after nerve crush). In contrast to the broad, bimodal distribution of single motor unit twitch rise times seen in normal muscles, motor units in reinnervated muscles yielded a narrower, unimodal distribution of rise times. Rise times of reinnervated units were intermediate to those of normal fast and slow units, suggesting that reinnervated units were composed of a mixture of fast and slow contracting fibers. An alternative possibility, that specific reinnervation was masked by contractile dedifferentiation of muscle fibers, was examined by maintaining a transmission blockade induced by botulinum toxin poisoning for an equivalent interval. Twitch rise times of treated motor units exhibited the distinctly bimodal distribution characteristic of normal muscles, suggesting that muscle fibers can retain contractile diversity during a transient period of denervation. We carried out computer simulations to estimate the amount of rise time diversity induced by varying degrees of specificity during reinnervation. Based on this analysis, we conclude that there is little if any selective reinnervation of muscle fiber types at the ages studied.

Animals↗
PubMed1989

Differential loss of neuromuscular connections according to activity level and spinal position of neonatal rabbit soleus motor neurons.

We have tested whether the ability of synapses to compete for occupancy of endplates during neuromuscular synapse elimination is affected by differences in the spinal position or in the activity level of the parent motor neuron. To test the role of spinal position, the relative sizes of motor units for motor neurons from middle and extreme (rostral/caudal) positions in the rabbit soleus motor pool were determined at 3 postnatal ages: 4-5 d ("early" ages, when the soleus is heavily polyinnervated), 8-9 d ("intermediate"), and 11-15 d ("late," when the soleus has just reached singly innervated state). Average motor unit sizes from extreme ventral roots were similar to those from middle ventral roots in early-aged soleus muscles but were significantly smaller (by 18-27%) for both intermediate and late muscles. Thus, motor neurons from extreme positions evidently compete less effectively for retention of synapses than those from middle positions. To test the role of differential activity, inactive and active synapses were pitted directly against one another by implanting Silastic plugs laden with tetrodotoxin (TTX) into one of the spinal nerves containing a minority of the soleus motor axons. Differential activity was maintained during a period of extensive synapse loss, from the time of the implant at day 4 or 5 until the intermediate age (day 8-9). Motor unit twitch tensions were subsequently measured to determine the relative number of synapses retained by individual active and inactive motor neurons. The inactivated motor units were on average significantly larger (by more than 50%) than the corresponding group from normal and control-implanted animals. The abnormally large size of inactivated motor units persisted in animals allowed to recover from the TTX block and examined after multiple innervation had disappeared. Hence, the effect of the TTX block cannot be attributed to a simple slowing of synapse elimination specifically among the inactive motor neurons. We conclude that complete presynaptic inactivity improves the chances of survival relative to that for normal activity during synapse elimination in the neonatal rabbit soleus muscle. This difference in competitive ability may contribute to the development of an important characteristic of adult muscles, the correlation between motor unit size and recruitment threshold.

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PubMedDate not supplied

Competition favouring inactive over active motor neurons during synapse elimination.

During normal postnatal maturation, mammalian muscles undergo an orderly process of synapse elimination, whereby each muscle fibre loses all but one of the multiple inputs with which it is endowed at birth. Experimental perturbations that increase or decrease the overall activity of nerve and/or muscle cause a corresponding increase or decrease in the overall rate of neuromuscular synapse elimination. On other grounds it has been suggested that competition among motor neurons is important in determining which synapses survive and which are eliminated. Would a difference in activity among the terminals at the same endplate affect the outcome of the competition and not just its rate? We investigated this issue by blocking activity for four days in a small fraction of the motor neurons innervating the neonatal rabbit soleus muscle. Twitch tensions of motor units were subsequently measured for both the active and inactive populations of neurons to assess whether the inactive neurons had lost fewer or more synapses than is normal. We found that inactive motor neurons have a significant advantage compared to active counterparts in control experiments, a finding opposite to that expected if the neuromuscular junction operated by classical 'Hebbian' rules of competition.

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